Knowledge of the molecular basis of protein-protein recognition is essential for understanding protein function, since the ability of proteins to form specific complexes with other proteins underlies most cellular processes. Our aim is to progress from purely anatomical descriptions of protein-protein interfaces to an understanding of how structural features contribute to the affinity and specificity of binding reactions. Dr. Mariuzza will carry out detailed structure-function studies of four distinct protein-protein recognition systems: 1) antigen-antibody, 2) natural killer (NK) cell receptor-MHC class I, 3) superantigen (SAG)-MBC class II, and 4) SAGT cell receptor (TCR). For each system, procedures have been developed for the expression of mutant proteins, the crystallization and structure determination of mutant complexes, and the measurement of thermodynamic binding parameters. The following aspects of the association process will be addressed: 1. Energetics of individual interactions across protein-protein interfaces. Double mutant cycles will be constructed to dissect coupling energies between residue pairs in antigen-antibody and NK receptor-MHC class I complexes. Unlike most interfaces described to date (including antigen-antibody), the NK receptorMHC class I interface is highly hydrophilic and dominated by charged interactions. It may represent a qualitatively different strategy that certain proteins have evolved for binding other proteins. 2. Structural basis for specificity in protein-protein recognition. The PI will determine the crystal structures of members of a family of antibodies that bind the immunizing antigen at coincident sites and with similar affinities, but which display striking differences in specificity towards a panel of antigenic variants. 3. Context dependence of the hydrophobic effect in protein-protein interfaces and the nature of energetic "hot spots." Using X-ray crystallography and titration calorimetry, the PI will examine the role that local environment and position in the interface play in determining the hydrophobic contribution of individual residues to binding. The results will improve our understanding of "hot spots" in protein-protein interfaces. 4. Structural determinants of affinity in the evolution of protein-protein interfaces. In order to clarify the structural mechanisms underlying affinity maturation, the PI will determine the crystal structures of antigenantibody, SAG-MHC class II, and SAG-TCR complexes that have acquired up to 10,000-fold increased affinities through directed evolution.